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蜜蜂克什米尔病毒(Kashmir bee virus,KBV)是一种高毒力的急性蜜蜂病毒,可以引起蜜蜂的死亡和蜂群崩溃。本研究旨在建立一种灵敏、快速检测KBV的TaqMan实时荧光RT-PCR检测方法。参照GenBank中polymerase polyprotein有关基因序列,设计一组特异性引物和探针,并通过体外转录法制备RNA标准品作为阳性模板。在对反应体系进行优化的基础上,建立了KBV的实时荧光RT-PCR检测方法并进行了特异性试验、敏感性试验、重复性试验和临床样本验证。结果显示:该方法能有效扩增8×100拷贝/μL～8×107拷贝/μL的KBV标准品,建立的标准曲线呈现良好的线性关系。该方法的检测灵敏度为8拷贝/μL,对其他蜜蜂病毒不发生交叉反应,具有很好的特异性;重复性试验结果显示组内和组间的变异系数分别低于1%和2%,重复性良好。应用本研究建立方法与常规RT-PCR方法对样品进行检测,TaqMan实时荧光RT-PCR检测方法的特异性优于常规RT-PCR。本研究建立的实时荧光RT-PCR检测具有良好的敏感性、特异性和重复性,为KBV的检测和流行病学调查提供技术支持。
Abstract：Kashmir bee virus(KBV) is an acute bee virus with high virulence which causes the death and collapse of the colony of honey bee. This study was conducted to establish a TaqMan based real-time RT-PCR for the detection of KBV. Based on the polymerase polyprotein gene sequence of KBV in GenBank,the primers and probe were designed,and the RNA standards was prepared as a positive template by in vitro transcription.A TaqMan based real-time RT-PCR assay was developed by optimizing the protocols of reaction,and the sensitivity,specificity and repeatability were evaluated,followed by clinical specimen verification. As results showed that the assay could detect the RNA standards between 8×100 copies/μL～8×107 copies/μL with a good linear relation,and the sensitivity limit was 8 copies/μL. There was no cross-reaction with other honey bee virus. The coefficient of variation(CV)for intra-assay and inter-assay repeatability was less than 1% and2% respectively. Samples were detected by real-time RT-PCR and common RT-PCR,and the results showed that the specificity of the former was much better than the latter. The assay for detecting KBV established in this study has a good sensitivity,specificity and repeatability,which can provide an effective means for detection and epidemiological investigation of KBV.
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基本信息:
DOI：10.13242/j.cnki.bingduxuebao.003937
中图分类号:S895
引用信息:
[1]张体银,邵碧英,林素洁,等.蜜蜂克什米尔病毒实时荧光RT-PCR检测方法的建立和评价[J].病毒学报,2021,37(03):678-685.DOI:10.13242/j.cnki.bingduxuebao.003937.
基金信息:
福建省对外合作项目(项目号：2018I0022),题目：7种常见蜜蜂病毒病快速检测技术研究~~
2021-02-08
2021
2021-03-04
2021-04-02
2021
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2021-04-06
2021-04-06
2021-04-06